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Allergenicity Assessment of Apple Cultivars: Hurdles in Quantifying Labile Fruit AllergensZuidmeer L.a · van Leeuwen W.A.a · Kleine Budde I.a · Breiteneder H.b · Ma Y.b · Mills C.c · Sancho A.I.c · Meulenbroek E.J.d · van de Weg E.d · Gilissen L.d · Ferreira F.e · Hoffmann-Sommergruber K.b · van Ree R.a
aDepartment of Experimental Immunology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; bDepartment of Pathophysiology, Center of Physiology and Pathophysiology, Medical University Vienna, Vienna, Austria; cInstitute of Food Research, Norwich, UK; dPlant Research International, Wageningen, The Netherlands; eDepartment of Molecular Biology, University of Salzburg, Salzburg, Austria
Background: Assessment of allergenicity of foods is important for allergic consumers and regulators. Immunoassays to measure major food allergens are widely applied, often giving variable results. Using the major apple allergen Mal d 1 as a model, we aimed to establish at the molecular level why different immunoassays for assessing allergenicity of apple cultivars produce conflicting outcomes. Methods: Mal d 1 was measured in 53 cultivars from Italy and 35 from The Netherlands, using four different immunoassays. Purified Mal d 1 standards were molecularly characterized by size-exclusion chromatography (SEC) and mass spectrometry (MS). Results: Three immunoassays using an identical standard gave similar results. Minor differences in sample preparation already resulted in significant loss of allergenicity. The fourth assay, using a different Mal d 1 standard, gave 10- to 100-fold lower outcomes. By SEC, this standard was shown to be almost fully aggregated. This aggregation was accompanied by a decrease of the mass of the Mal d 1 molecule by ∼1 kDa as analyzed by MS. The deviating immunoassay was shown to selectively recognize this aggregated form of Mal d 1, whereas the other three assays, including the one based on IgE antibody recognition, preferentially bound non-aggregated allergen. Conclusions: Variable and poorly controllable major allergen modification in both extracts and standards hamper accurate allergenicity assessments of fruits.
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